1. Field of the Invention
This invention relates to the field of therapeutic methodologies for treating viral infections. More particularly, this invention provides a method for reducing the viral load by extracorporeal treatment of a patient""s blood with immobilized molecules having specific affinity for viral components.
2. Description of Related Art
Human immunodeficiency virus (HIV) is the etiological agent of acquired immunodeficiency syndrome (AIDS) and infects selected cells of the immune system thereby compromising the infected individual""s immune response. It is estimated that there are over 1 million HIV infected individuals in the United States and over 13 million world-wide. The clinical course of HIV infection typically consists of a prolonged asymptomatic state, followed by a depletion of T4 lymphocytes making the individual susceptible to opportunistic infections and neoplasms.
Currently there is no cure available for HIV infection. Mononucleoside drugs like AZT, dI, ddC and d4T, which inhibit the reverse transcriptase, have been approved for the treatment of HIV. Further, proteases inhibitors are also being now used. However, the emergence of drug resistant mutants limit the usefulness of these drugs.
The development of an effective vaccine against HIV infections has been hampered, in part, due to the rapid mutation of the HIV genome, and due to inaccessibility of immunogenic epitopes of viral proteins. The entire HIV genome has now been sequenced (Ratner et al., 1987, AIDS Res. Hum. Retroviruses, 3(1):57-69, incorporated herein by reference). The HIV genome encodes three major structural genes, gag, pol and env, which are flanked at either end by long terminal repeat (LTR) sequences. The HIV genes encode 3 viral enzymes, reverse transcriptase (p51+p66+RNase H), integrase (p32), and protease (p12), encased in a cylindrical protein core, composed predominantly of p24. Matrix protein (p17) and a lipid membrane, which contains two major envelope glycoproteins, gp41 and gp120, surround the protein core. The virion has a diameter of 110 nm (Gallo, 1995, Nat. Med., 1:753-759).
In order for HIV-1 to infect a cell, viral gp120 must bind to CD4 as well as to a surface chemokine receptor, generally CCR5(R5) or CxCR4(X4). Once endocytosed, the virion is uncoated. The viral RNA is reverse transcribed into double-stranded DNA, enters the cell nucleus and integrates into the host genome. Transcription of the integrated viral DNA results in the production of viral RNA, as well as various mRNA which are translated into viral proteins, some of which require further proteolytic processing. Mature virions are assembled and released from the cell by budding. (Fauci et al., 1996, Ann. Int. Med., 124:654-63). A dying cell may also release all its contents including intact virions, and fragments thereof into the blood. Thus, circulating blood of HIV-infected individuals contains both intact virions, fragments thereof and free RNA.
Isolates of HIV-1 from various donors show variability in surface glycoproteins. The variability in gp120 is more than the variability in gp41. Five variable regions are interspersed with conserved sequences within gp120. The majority of neutralizing antibodies to HIV-1 is directed towards the V3 loop of gp120, although some neutralizing antibodies also recognize the V2 and C4 domains, as well as epitopes in gp41. Within the V3 loop of gp120, the most highly conserved subdomain is glycine317-proline318-glycine319-arginine320-alanine321-phenylalanine322. Viruses with mutations involving glycine317-proline318-alanine319 are not infectious. Envelope sequences divides, HIV-1 into M and O groups. Within the M subgroups A-H are recognized, with subgroup B being prevalent in the United States.
HIV-1 replication occurs predominantly in CD4+lymphocytes, the majority of which are located in lymphoid organs, such as peripheral lymph nodes and spleen. HIV-1 can also be found in macrophages and macrophage-like cells, such as microglia in the central nervous system (Cohen et al., 1997, Immunol Rev. 159:31-48). Plasma HIV-1 levels and presence of HIV-1 infected lymphocytes in peripheral blood strongly correlate with the clinical status of HIV-1 infected patients (Ferre et al., 1995, J Acquir. Immnune Defic. Syndr. Hum. Retrovirol 10:S51-6; Obrien et al., 1996, N Engl J Med., 334:426-431). Half-life of circulating virions is 6 hours, while the half-life of HIV-1 infected cells in peripheral blood is 1.6 days. Greater than 1010 virions may be released into the circulation each day (Ho et al., 1995, Nature 373:123-126; Wei et al., 1995, Nature 373:117-122.). The ability of the host immune system to keep HIV infection in check, and limit clinical symptoms, is directly proportional to the viral burden. Anti-retroviral therapies, nucleoside analogues, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors, aim to reduce the viral burden so that the immune system can control or clear residual infection (Fauci et al., 1998, Harrison""s Principles of Internal Medicine, p. 1791-1856).
No previous technologies have been developed which directly absorb HIV from the blood using in vivo dialysis or immunoabsorption. Extracorporeal perfusion of plasma over protein A, plasmapheresis and lymphapheresis have all been used as immunomodulatory treatments for HIV infection, and the thrombocytopenia resulting from it (Kiprov, 1990, Curr. Stud. Hematol. Blood Transfus., 57:184-97; Snyder et al., 1989, Artif Organs 13:71-7; Mittelman, 1989, Semin Hematol 26:15-8; Snyder et al., 1991, AIDS 5:1257-60.). These therapies are all proposed to work by removing immune complexes and other humoral mediators which are generated during HIV infection. They do not directly remove HIV virus. Extracorporeal photopheresis has been tested in preliminary trials as a mechanism to limit viral replication (Bisaccia et al., 1990, Intern Med 113:270-275; Bisaccia et al., 1993, J. Acquir Immune. Defic. Syndr. 6:386-92). It does not absorb virus from the blood. It has been reported that rabbit antisera raised against HIV proteins, when coupled to Sepharose 4B or Silica, could be used for extracorporeal removal of HIV proteins from the blood of rabbits which had been injected with recombinant HIV proteins (Lopukhin et al., 1991, Vestn Akad Med Nauk SSSR 11:60-3). This strategy was inefficient, and apparently not pursued. It required extracorporeal absorption of blood, and did not provide for a mechanism to remove free HIV viral particles from the blood (Lopukhin et al., 1991, supra).
Thus, there is an ongoing need for novel therapeutic approaches to the treatment of HIV and other viral infections. In particular, there is a need for the development of novel approaches to reduce the viral load so as to increase the effectiveness of other treatments and/or the immune response.
The present invention provides a method for reducing the viral load of patients. The method of the present invention involves removal of intact virions, fragments or components thereof including free viral nucleic acid from the blood by extracorporeally circulating blood through hollow fibers which have in the porous exterior surface, affinity molecules having specificity for viral components. Passage of the blood through the hollow fibers causes the virions and components thereof to bind to the affinity molecules thereby reducing the viral load in the effluent.
In one embodiment, this invention uses DNA sequences which have been shown to hybridize to many isolated subtypes of HIV-1 in vitro (Wilber, 1997, supra), and antibodies which have been shown to bind to intact virions of many HIV-1 subtypes in vitro (VanCott et al., 1994, Immunol. 153:449-59).
The method of the present invention reduces the number of virions in the blood, and thus contributes significantly to the reduction of viral load. It will be apparent to those skilled in the art that the device can be modified to assist in the clearance of other viral infections, frequently occurring simultaneously with HIV-1, such as cytomegalovirus (CMV), hepatitis B virus (HBV), and hepatitis C virus (HCV) (Fauci et al., 1998, supra).
Thus, an object of the invention is to provide a method for reducing the viral load in the blood of an individual infected with the virus.
Another object of the present invention is to provide a method for reducing the viral load in the blood by extracorporeal circulation of blood through hollow fibers containing immobilized molecules having specific affinity for viral components.
Another object of the present invention is to provide an apparatus comprising hollow fibers, wherein the exterior surface of the fibers is in close proximity with molecules having specific affinity for target molecules in the virus.